Bottom Line:
Unexpectedly, short interfering RNA designed to deplete CRACR2A had no effect on CRAC channels in endothelial cells but reduced the abundance of a protein with about twice the mass of CRACR2A.It made a positive contribution to endothelial tube formation.The data suggest that endothelial cells contain a long variant of CRACR2A which is an EF-hand-containing Rab protein that lacks impact on CRAC channels.

f0010: Detection of long but not short CRACR2A in endothelial cells. (A) Western blot probed with anti-CRACR2A antibody for HUVECs transfected with control or CRACR2A siRNA. S indicates the expected mass for CRACR2A (short variant). L indicates a larger protein (long variant) which was depleted by CRACR2A siRNA. Equal total protein was loaded in each lane. (B) Densitometry analysis for the large protein (L) seen in blots of the type shown in (A). Control siRNA was compared in paired experiments with CRACR2A siRNA 1 or CRACR2A siRNA 2 (n = 3 each). The band intensity in the CRACRA siRNA group is normalized to that in its control siRNA group. Equal total protein loading in each lane was validated by using anti-β-actin antibody. (C) PCR products with (+) or without (−) reverse transcriptase (RT) and using primers to the extended 3′ sequence of CRACR2A-L. Reactions are shown for mRNA isolated from human endothelial cells from pulmonary artery (HPAEC), dermal blood (HDBEC), umbilical artery (HUAEC), colon microv asculature (HCoMEC), cardiac microvasculature (HCMEC), pulmonary microvasculature (HPMEC), dermal microvasculature (DMEC) or bladder microvasculature (HBdMEC). The expected product was 214 bp.

Mentions:
An explanation for the above data could be that CRACR2A is not expressed. Indeed, anti-CRACR2A antibody failed to detect protein of ∼45 kDa (Fig. 2A), which is the expected mass of CRACR2A [3]. However, a doublet around 95 kDa was labelled by the antibody and the lower of these two bands was depleted by two different siRNAs targeted to CRACR2A (Fig. 2A and B). The upper band may reflect an unrelated protein, labelled non-specifically by the antibody. The data suggest that CRACR2A is expressed in endothelial cells but that it occurs at about twice the expected molecular mass.

f0010: Detection of long but not short CRACR2A in endothelial cells. (A) Western blot probed with anti-CRACR2A antibody for HUVECs transfected with control or CRACR2A siRNA. S indicates the expected mass for CRACR2A (short variant). L indicates a larger protein (long variant) which was depleted by CRACR2A siRNA. Equal total protein was loaded in each lane. (B) Densitometry analysis for the large protein (L) seen in blots of the type shown in (A). Control siRNA was compared in paired experiments with CRACR2A siRNA 1 or CRACR2A siRNA 2 (n = 3 each). The band intensity in the CRACRA siRNA group is normalized to that in its control siRNA group. Equal total protein loading in each lane was validated by using anti-β-actin antibody. (C) PCR products with (+) or without (−) reverse transcriptase (RT) and using primers to the extended 3′ sequence of CRACR2A-L. Reactions are shown for mRNA isolated from human endothelial cells from pulmonary artery (HPAEC), dermal blood (HDBEC), umbilical artery (HUAEC), colon microv asculature (HCoMEC), cardiac microvasculature (HCMEC), pulmonary microvasculature (HPMEC), dermal microvasculature (DMEC) or bladder microvasculature (HBdMEC). The expected product was 214 bp.

Mentions:
An explanation for the above data could be that CRACR2A is not expressed. Indeed, anti-CRACR2A antibody failed to detect protein of ∼45 kDa (Fig. 2A), which is the expected mass of CRACR2A [3]. However, a doublet around 95 kDa was labelled by the antibody and the lower of these two bands was depleted by two different siRNAs targeted to CRACR2A (Fig. 2A and B). The upper band may reflect an unrelated protein, labelled non-specifically by the antibody. The data suggest that CRACR2A is expressed in endothelial cells but that it occurs at about twice the expected molecular mass.

Bottom Line:
Unexpectedly, short interfering RNA designed to deplete CRACR2A had no effect on CRAC channels in endothelial cells but reduced the abundance of a protein with about twice the mass of CRACR2A.It made a positive contribution to endothelial tube formation.The data suggest that endothelial cells contain a long variant of CRACR2A which is an EF-hand-containing Rab protein that lacks impact on CRAC channels.